Tissue‐engineered tendon nano‐constructs for repair of chronic rotator cuff tears in large animal models

Abstract Chronic rotator cuff tears (RCTs) are one of the most common injuries of shoulder pain. Despite the recent advances in surgical techniques and improved clinical outcomes of arthroscopically repaired rotator cuffs (RCs), complete functional recovery—without retear—of the RC tendon through tendon‐to‐bone interface (TBI) regeneration remains a key clinical goal to be achieved. Inspired by the highly organized nanostructured extracellular matrix in RC tendon tissue, we propose herein a tissue‐engineered tendon nano‐construct (TNC) for RC tendon regeneration. When compared with two currently used strategies (i.e., transosseous sutures and stem cell injections), our nano‐construct facilitated more significant healing of all parts of the TBI (i.e., tendon, fibrocartilages, and bone) in both rabbit and pig RCT models owing to its enhancements in cell proliferation and differentiation, protein expression, and growth factor secretion. Overall, our findings demonstrate the high potential of this transplantable tendon nano‐construct for clinical repair of chronic RCTs.

tendon region, which is composed of well-aligned collagen I fibers; an uncalcified fibrocartilage zone; a calcified fibrocartilage zone; and the bone region, which is composed of collagen type I. 10 The regions forming this gradient structure are continuous, with no clearly defined borders in between, and they are not clearly distinguishable even at the ultrastructural level. 11 This unique structure is responsible for distributing mechanical stress and enhancing the bonding strength between soft and hard tissues. Because of this structure, full functional recovery of the RC cannot be achieved without regeneration of the TBI. In fact, incomplete TBI regeneration after arthroscopic repair is responsible for the high retear rates of up to 27%-94%. 2 Therefore, a new strategy is needed to aid complete TBI regeneration.
Biological scaffolds have emerged as a promising alternative to current arthroscopic repair. Currently, collagen-based biological scaffolds (e.g., GRAFTJACKET™ by Geistlich) are most commonly used for the clinical repair of RCTs. This type of scaffold contains extracellular matrix (ECM) structures typically extracted from sources such as the porcine intestinal submucosa, porcine dermis, human fascia, and human dermis. Although collagen is a biomaterial that clinical physicians worldwide are most familiar with, collagen-based biological scaffolds have not shown promising clinical outcomes for RCT repair owing to their low functionality, high swelling rate, bioresorbability, low mechanical support of the RC tendon, and high costs over the past few years. [12][13][14][15] To overcome these limitations, researchers have fabricated biomimetic scaffolds based on the synthetic biodegradable polymers, such as polycaprolactone (PCL), poly(lactic-co-glycolic acid), and polylactic acid, to mimic the ECM of the RC tendon as an alternative strategy for tendon tissue regeneration. [16][17][18][19] Peach et al. reported the regeneration effect of an electrospun PCL scaffold that mimicked the RC tendon tissue microenvironment in a rat model. 20 Inspired by the high alignment and well-defined organization of the ECM of natural tendons, our group also developed a PCL-based nanotopographic scaffold for RC tendon regeneration. 19 However, despite rapid advances in the development of biodegradable biomimetic scaffolds, the clinical efficacy of a scaffold-only strategy for RC tendon regeneration is still questionable, as many in vivo studies have not shown a significant effect with this method. Furthermore, to the best of our knowledge, there are very few clinical trials and no reports on the clinical efficacy of biodegradable biomimetic scaffolds.
Recently, stem cell-based strategy has received tremendous attention for promising candidates for RCT regeneration. [21][22][23][24] The key factors of which are the regeneration of the TBI through the direct differentiation of stem cells into bone, tendon, cartilage, and ligament (i.e., the structures of the TBI) and the autocrine and paracrine effects of growth factors and cytokines from the stem cells. Kim et al. demonstrated that the application of adipose-derived mesenchymal stem cells (MSCs) after surgical repair of an RCT could improve the shoulder functions. 22 In a first-ever human clinical trial, Jo et al.
injected adipose-derived MSCs into the RC of patients; although the therapy improved the shoulder functions slightly and relieved pain, it did not directly promote RC regeneration. 25 Despite the slightly positive clinical outcomes, stem cell-based therapy still has the following critical limitations: (i) the efficiency of stem cell transplantation into the injury site is very low (i.e., the loss rate due to injection is high) and (ii) the injected stem cells fail to achieve stable adhesion and continuous proliferation in the RC tendon, resulting in limited success in repair of the tear. [26][27][28] Therefore, there is a need for a functional platform or construct that enables integration of the transplanted stem cells into the tissues and provides an environment that promotes continuous tissue regeneration.
As described earlier, the TBI of RCs consists of a structural and compositional gradient integrated through fibrocartilaginous junctions. 29,30 Because of its hierarchical and complex structure, functional regeneration of the TBI is restricted and thus considered a great clinical challenge. An ideal construct for RCT repair should provide an environment conducive to overcoming the restricted healing capacity of the TBI. The transplantable construct should also exhibit sufficient mechanical properties for supporting the RC tendon as well as allow for the control of biodegradability and cell functions to enable functional TBI and thereby RC tendon regeneration. 31,32 With these key considerations in mind, we propose herein a novel tissue-engineered tendon nano-construct (TNC) composed of human mesenchymal stem cells (hMSCs) on a scaffold that mimics the highly aligned nanotopographic structures of the ECM of native tendon tissue. Using capillary force lithography, we first fabricated a US Food and Drug Administration-approved PCL-based biomimetic scaffold bearing a nanotopographic surface possessing highly aligned ECM-like structures. The TNC was then developed by incorporating hMSCs onto the fabricated scaffold. Next, we investigated the influence of the nanotopographic cues on the hMSCs effects on RC tendon regeneration in rabbit and pig RCT models. To verify the in vivo regeneration effects of the TNC, we also conducted in vitro experiments to investigate the influence of the scaffold's nanotopographic cues on the morphology, adhesion, proliferation, and mineralization of the hMSCs as well as the expression of proteins related to tenogenic differentiation and tendon regeneration and secretion of growth factors by these cells.

| Characteristics and transplantation of tissue-engineered tendon nano-constructs
A schematic of the RCT repair strategy of TNC transplantation is shown in Figure 1a. Scanning electron microscopy (SEM) images of the surface morphologies of a flat scaffold and a tendon ECM-inspired scaffold, as well as cross-sectional SEM images, revealed the former construct to have a flat surface and the latter to have a highly aligned topography with grooves and ridges (~800 nm size) similar to those of the highly aligned structure of the native tendon ECM (Figure 1b,c).
The 3D structures of these two scaffolds were confirmed using atomic force microscopy ( Figure 1d Table S1). An assessment of the scaffold wettability through measurement of the water contact angle showed that the value for the nanotopographic scaffold (58.17 ± 2 ) was lower than that for the flat scaffold (78.3 ± 2 ). In our previous report, we confirmed that the contact angle was larger in the direction of parallel to the nanogroove than in the direction of perpendicular to the nanogroove. In this work, the contact angle was measured parallel to the nanogroove ( Figure 1j). The formation of aligned tendon tissue was observed on the nano scaffolds cultured hMSCs. To characterize the hMSCs used in this study, their expression of specific surface markers was analyzed using fluorescence-activated cell sorting. The hMSCs at passage 3 showed positive CD105, CD90, and CD44 expressions and a negative CD45 expression (Figure 1k).

| Tendon regeneration effect of tissue-engineered tendon nano-constructs in rabbit animal model
Our in vivo study demonstrated that the TNC could promote RC tendon regeneration, indicating that this construct contributes the following three clinically important advances in RCT repair: (i) the ability to evaluate RC tendon regeneration in large animal models, (ii) effective healing of the TBI junctions; and (iii) bone healing via the cascade effect. First, we hypothesized that the TNC with a highly aligned structure that mimics the native RC tendon would promote TBI regeneration (including the RC tendon, fibrocartilages, and bone).
To prove our hypothesis, the TNC was tested in both rabbit and pig models of chronic RCT. We then compared the performance of three Specifically, the rate of collagen I deposition was in the order of suture therapy (13%) < stem cell therapy (21%) < TNC therapy (26%) ( Figure 2c). To quantify these histological results, semi-quantitative scoring was performed using the Bonar scoring system ( Figure 2d and Table S2). Both the TNC and stem cell therapy groups had significantly higher scores than that of the suture group, with the values in the TNC therapy group being the most statistically significantly.

| Tendon regeneration effect of tissueengineered tendon nano-constructs in large animal model
To confirm the regenerative effect of the TNC in a larger animal, the entire evaluation was repeated in a pig model of RCT. Figure 3a depicts the patch transplantation process, and Figure 3b shows images of native and ruptured RC tendon tissues. Interestingly, the compact and aligned tendon tissue was formed below the TNC insertion compared with hMSCs only (Figure 3c). Importantly, the TNC therapy again resulted in the well-organized, highly aligned, and highly dense collagen fibers to the greatest extent, as is evident in the H&E and M&T staining images ( Figure 3d). In general, the safranin-Ostaining is performed to confirm the mineralized fibrocartilage regeneration, we also confirmed the mineralized fibrocartilage regeneration by the staining. The staining images show a higher mineralized fibrocartilage regeneration in our TNC therapy compared to suture and stem cell therapy in both rabbit and pig model (Figures 2 and 3d). For the more accurate comparison of mineralized fibrocartilage regeneration, we have added the quantification data of stained area ( Figure S2). Furthermore, to confirm the mineralization of hMSCs, we performed the osteogenic mineralization of hMSCs on the nano scaffolds. The quantification of the osteogenic mineralization on the flat and nano scaffolds demonstrated the highest degree of osteogenic mineralization by the hMSCs on the nano surface. In previous studies, the correlation between the osteogenic mineralization and fibrocartilage regeneration has been reported. These in vivo and in vitro results demonstrated that the proposed TNC therapy promotes the mineralized fibrocartilage regeneration. Also, these results again indicate that the precisely aligned nanotopography and stem cells could synergistically promote TBI regeneration not only in rabbits but also in pigs, whose RC tendon is known to be similar to that of humans. As in the rabbit study, Picrosirius red staining of the regenerated pig tissue was performed, whereupon the highest total collagen deposition along with a significantly improved collagen I percentage was shown in the F I G U R E 2 Effects of the tissue-engineered tendon nano-constructs on rotator cuff (RC) tendon regeneration in rabbit animal model.  Table S2). Therefore, these successful outcomes of TBI regeneration in both the rabbit and pig models of RCT suggest the clinical applicability of the proposed TNC.

| Analysis of bone formation and biomechanical properties of regenerated tendon tissue
In the evaluation of RCT repair techniques, the functional evaluation of the regenerated RC tissue is as important as histological evaluations. In this study, micro-computed tomography (CT) and biomechanical tests were used for the functional evaluation of the regenerated RC tendon.
Reconstructed micro-CT images of the proximal humerus were used to determine the morphologies of the regenerated bone in both the rabbit and pig models (Figure 4a). The tendon-bone insertion site of the proximal humerus showed more significant bone formation after TNC implantation than after suturing or stem cell injection. The tissues repaired using the TNC showed increases in their total volume and bone mineralization density when compared with those repaired with the other methods in both animal models (Figure 4b,c). These results suggest that our TNC has a remarkable capacity for enhancing new bone formation at the TBI, with a potentially higher success rate in healing RCTs than that of currently used methods. Furthermore, at 6 weeks, the cross-sectional area of the TNC-repaired tendon tissue was higher (~221 mm 2 ) than that of the stem cell-repaired (~202 mm 2 ) and suturerepaired tendon tissues (~148 mm 2 ) ( Figure S1A) and the ultimate stress was higher (~78.8 MPa) than that of the stem cell-repaired (~67.4 MPa) and suture-repaired tendon tissues (~48.1 MPa) ( Figure S1B). Also, ten- 2.5 | Cell morphological response to tissueengineered nano-constructs for tendon regeneration

| Effects of tissue-engineered nano-constructs cues on stem cell functions
Although there was no significant difference in hMSCs attachment on the two types of scaffolds, cell proliferation was more than 10% higher on the ECM-inspired scaffold after 3 days (Figure 5c To verify whether the nanotopographic cues could affect the expression of proteins related to the induction of the dense and wellorganized RC tendon tissue and tenogenic differentiation of the hMSCs, western blot analysis was carried out to detect those specific proteins. Collagen type I, scleraxis, tenomodulin, and decorin, which play important roles in tenogenic differentiation and tendon regeneration, were found to be upregulated in the cells on the nanotopographic scaffold relative to their expression in cells on the flat scaffold. In contrast, collagen type III was slightly downregulated on the ECM-inspired scaffold (Figure 5f,g). It is known that stem cells generate autocrine and paracrine factors, which are crucial molecules for signaling the activation of various cellular functions. The secretion of growth factors by hMSCs cultured on the two types of scaffold was analyzed. The hMSCs on the ECM-inspired scaffold secreted higher levels of basic fibroblast growth factor, bone morphogenetic protein (BMP)-4, BMP-7, epidermal growth factor, epidermal growth factor receptor, fibroblast growth factor (FGF)-4, FGF-7, glia cellderived neurotrophic factor, hepatocyte growth factor, osteoprotegerin, transforming growth factor (TGF)-alpha, TGFβ1, TGFβ3, and insulin-like growth factor-1 (Figure 5h).
In conclusion, our results have demonstrated that our rational design of a tissue-engineered TNC has great potential for use in the repair of chronic RCTs, as it facilitated functional tendon regeneration in large animal models, the findings of which were supported by in vitro test results. We propose that this new TNC therapy could replace the current surgical methods used for RCT repair and improve shoulder functions through its healing of the TBI.
The medium was changed every 3 days. All stem cells used in this work were at passage 3-5.

| Design and fabrication of a nanoengineeredartificial stem cell construct
The detailed method to fabricate PCL-based tendon-inspired scaffold has already been reported by our group. Briefly, a thin PCL scaffold was fabricated by spin coating the PCL solution that was poured into glass on the vacuum cuck of the spin coater. After the thermal imprinting process, the assembly of the PCL layer on circular glass and PDMS molds was cooled at 25 C for 30 min. The fabrication process of the tendon-inspired scaffold is following which PDMS molds were used to create either a flat or a nanotopographical (800 nm, ridges, and grooves) pattern on the melted film surface under pressure at 80 C for 2 min.

| Characteristics and properties analysis
The tissue-engineered nano-constructs were analyzed using high-res-

| Mechanical properties analysis
The mechanical tests of the all scaffolds were performed using MCT-1150 tensile testers (A&D Company, Japan) at a test speed of 100 mm/min. The tests included the analysis of 10 specimens per sample with the same interval set. Normal and shear adhesion forces of all scaffolds were evaluated using porcine rind and measured using an MCT-1150 instrument at a test speed of 50 mm/ min. Prior to the adhesion test, a fresh porcine rind was rinsed with deionized water, all scaffolds were attached to its surface, and rind/scaffolds measured under a preload of~0.5 cm 2 . The pulling weight was gradually increased until the adhesion force felled off.

| Western blotting
To confirm the protein expression levels of collagen III, collagen I, scleraxis, tenascin C, TNMD, and decorin, hMSCs were first cultured on the tendon inspired scaffolds for 12 h, following which 1 Â 105 cells/sample were cultured in medium (Promo Cell) for 7 days. Thereafter, the cells were washed twice with cold PBS and lysed by ultrasonication in RIPA buffer for 30 min at 4 C. The lysates were collected by centrifugation at 12,000 Â g for 15 min at 4 C. After washing, the cells twice with cold PBS, they were lysed again with a modified radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.

| Growth factors array
To ascertain whether the hMSCs could secrete growth factors and cytokines on the flat and nanotopographical scaffolds, the hMSCs

| Histological observations and evaluation
The proximal humerus including the greater tuberosity head with attached supraspinatus tendon of both shoulder of each rabbit was harvested. Specimens were fixed in neutral buffered 10% formalin (pH 7.4) and decalcified with Calci-Clear Rapid (National Diagnostics, Atlanta) for 2 weeks, and paraffin blocks were made in the repair site including supraspinatus tendon and greater tuberosity. Sections (4 μm thickness) were cut in the coronal plane and stained with hematoxylin and eosin (H&E) and Masson's trichrome. We assessed cellularity, collagen fiber continuity, orientation, density, and maturation of the tendon-to-bone interface, and we also evaluated the inflammation rate around patch at the tendon-to-patch interface. Images were captured and acquired using an Aperio Image Scope (Leica, Ca, USA) software. General histological evaluation was performed with hematoxylin and eosin (200Â magnification), Masson's trichrome (200Â magnification), and Picrosirius red (100Â magnification) stained slides of chronic RC tear animal models. The slides were evaluated using the semiquantitative grading scale of Bonar score, which assess four variables (cell morphology, ground substance, collagen arrangement, and vascularity) of tendon to bone interfaces. A four-point scoring system is used, where 0 indicates a normal appearance and 3 a markedly abnormal appearance (Table S1). The total histological scores for each group were calculated from the sum of these four characteristic grades. Four sections were randomly selected form each group and were evaluated blindly by three independent assessors.
The average score was used for comparison. BV), bone mineral density (BMD) was calculated for a ROI located at the greater tuberosity. At 6 weeks after surgery, 18 animals were sacrificed for biomechanical testing (four rabbits per group per time point). The humerus with attached supraspinatus tendon was dissected from the surrounding tissues. For the biomechanical testing, the harvested tissues were wrapped in saline-soaked gauze and kept at À80 C. Before testing, the tissues were thawed with saline wet gauze at room temperature for 24 h and the tissues were kept moist with saline during all of the tests. The proximal end of the tendon was compressed with sandpaper, gauge, and rubber to prevent slippage and to reduce damage to the specimens. The complex was clamped vertically in the custom-designed upper jig. Testing was performed with the shoulders at 90 of abduction with a material testing system (H5K5; Tinus Olsen, England, UK). All specimens were initially preloaded to 0.2 N and preconditioned for five cycles under 5% of strain at a rate of 0.1 mm/s. Then, these specimens were loaded to failure in tension at a constant rate of 0.1 mm/s. The cross-sectional area of the supraspinatus tendon was measured at the TBI. The loaddisplacement curve recorded during tests and the tensile stress, load to failure, ultimate stress, stiffness, and elastic modulus were calculated. In detail, Young's modulus is calculated following formula:

| Analysis of RC tendon healing by microcomputed topography and biomechanical test
Young's modulus = stress/strain and stiffness are calculated following formula: stiffness = Young's modulus * area/length.

CONFLICT OF INTEREST
Jangho Kim is named on patents that describe the use of stem cells platforms for tissue regeneration. All other authors declare that they have no competing interests.

DATA AVAILABILITY STATEMENT
All data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. Additional data related to this paper may be requested from the corresponding author on reasonable request.